Kit-Free Site Directed Mutagenesis Protocol

Molecular biologists are familiar with the QuikChange® Site-Directed Mutagenesis Kit that allows rapid intoduction of a point mutant into a plasmid/vector/mammalian expression construct. Briefly, the protocol first involves a thermocycling/PCR step with mutagenic primers, followed by a DpnI digestion step to digest the methylated parental/wild-type plasmid, and finally transformation into competent cells for nick repair.

An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

Most experts we’ve talked to still use this technique, but don’t see the point of an expensive kit. Instead they use their own protocols with inexpensive enzymes and reagents bought separately. One such protocol involves the following:

Step 7: Load 10ul of completed reaction mixed with SafeGreen™ Loading Dye next to 1kb DNA ladder and run the gel at 150V for 10 min to determine if PCR was successful. You should not see a product for the ‘negative control’ tube that had no polymerase. Troublshooting: If PCR reaction yields no product, then do gradient for the annealing temperature. The DMSO is extremely important, so keep this in the reactions.

Step 8: Mix PCR reaction with 500ul of PB buffer and purify on column. Wash with PE buffer and elute with 50 ul of water. Use 10ul of for transformation of TransforMax™ EC100™ competent cells.

Interested in using this protocol to your lab? Here is a list of products you will need: